Small molecules that bind to monomeric or filamentous actin elicit their antiproliferative effects by impairing the ability of cells to progress through the cell cycle and undergo cytokinesis due to the defective actin cytoskeleton. Understanding the mode of action of such compounds expands our knowledge of actin biochemistry and provides opportunities for the development of new therapeutic agents. Traditionally, studies of the mechanism of action of small-molecule modulators of actin polymerization entailed the investigations of changes in fluorescence intensity during polymerization or depolymerization of pyrene- or prodan-labeled actin in solution. However, the main limitation of this method arises from the fact that the changes in the fluorescence intensity may reflect the binding of a small molecule to the filaments, especially if the binding event occurs in the close proximity to the dye, resulting in quenching of fluorescence, which could be independent of the rates of actin depolymerization. Total internal reflection fluorescence (TIRF) microscopy has recently emerged as a powerful tool to investigate actin filament dynamics and its regulation by several actin-binding proteins, as well as latrunculin A, which is incapable of depolymerizing filamentous actin in vitro. We used TIRF microscopy to directly observe actin filament severing by a series of small molecules, which are derived from bistramide A—a marine natural product that specifically and potently targets actin in the cell (Statsuk, et al. (2005) Nat Chem Biol 1:383-388; incorporated by reference). In addition, we demonstrated that the C(1)-C(4) enone-containing subunit of this natural product plays a pivotal role in covalent modification of cellular actin, which further enhances the cytotoxicity of the corresponding bistramide-based compounds. Our study provides comprehensive characterization of the unique mode of action of bistramide A and identifies structural requirements of bistramides that are responsible for severing actin filaments and inhibiting growth of cancer cells in vitro and in vivo (Gouiffe's, et al. (1998) Tetrahedron 44:451-459; Biard, et al. (1994) J Nat Prod 57:1336-1345; Riou, et al. (1993) Anticancer Res 13:2331-2334; Rizvi et al. (2008) PNAS 105: 4088-4092; the entireties of all of which are hereby incorporated by reference).